Process for preparing biotin

ABSTRACT

Biotin is prepared by (a) ozonizing methylcyclohexene; (b) reacting nitromethane with the methyl-6-oxohexanoate from a to form 7-nitro-6 hydroxyheptanoic acid methyl ester; (c) acylating to form the corresponding 6 acyloxy compound; (d) forming a double bond at the 6 position; (e) reacting the product of d with nitroethanethiol to form dl-7-thia-6-nitromethyl-9-nitro-nonanoic acid methyl ester; (f) forming a furoxan by cyclization; (g) reducing the furoxan and acylating to form dl-2(4-carbomethoxybutyl)-3,4-bis(acylamido)-2,5-dihydro thiophene; (h) selectively hydrogenating to form the 3,4-cis-bis(acylamido)-2,5-dihydro thiophene; and (i) hydrolyzing and cyclizing to form dl-biotin. The products formed at steps (e)-(h) are novel, as are the individual steps (e), (f), (g), and (h).

BACKGROUND OF THE INVENTION

1. Field of the Invention

This invention relates to a unique total synthesis for preparing dlbiotin which starts with methoxycyclohexene and utilizes the basicconcept of forming a furoxan system to construct the biotin nucleus.Within the total synthesis of biotin are several unique individual stepsheretofore unknown in the prior art, as well as several uniqueintermediates which are formed in the process for making biotin.

2. Prior Art

Biotin is one of the water soluble vitamins which is a monocarboxylicacid containing a cylic urea structure with a sulfur in a thioetherlinkage as shown in FIG. 1 below ##STR1## Biotin contains 3 asymmetriccarbons (circled, above) and therefore there can exist as 4 racemates or8 stereo isomers of the biotin structure. As a member of the vitamin Bfamily biotin has, for a long time, been also known as the essentialfactor in the processes and maintenance of normal metabolism. Biotin'sroles are discussed in an article entitled "Biotin-A Ubiquitous AndVersatile Vitamin" by Dr. J. C. Bauernfeind, Feed Stuffs, 41, 32-34,1969.

Several processes are known for preparing biotin and these are discussedin Comprehensive Biochemistry, 11, 66-81, Chapter VI entitled "Biotin"by L. H. Sternbach. As pointed out in that discussion, biotin was firstsynthesized by Harris and co-workers in the early 40's by forming athiophene intermediate and eventually forming the keto 3,4 imidazolidogroup as the last step. A second route started with thiophane andproceeded through a series of reactions finally forming the 3,4imidazolido group at the end of the reaction. A third synthesis alsostarted with a substituted thiophane and progressed in a way similar tothe second synthesis, however at each step meticulous care was taken toestablish the steric configuration of the substituents in positions 3and 4.

The commercial synthesis of biotin was developed by Hoffman La Roche anddiffers from the first three syntheses in several respects, i.a. thatthe imidazolidone ring is formed first with the 2 constituents(carbonyls) cis to each other.

The process for the synthesis of biotin according to this invention is acomplete departure from anything taught in the prior art and involvesstarting with an alkoxy cyclohexane and proceeding through various stepsto form a novel thieno furoxan structure and novel thiophenes toultimately form biotin.

SUMMARY OF THE INVENTION

The process for preparing biotin by the process of this invention is setforth in the following reaction scheme ##STR2## wherein R and R¹ may bealkyl of 1-4 carbons and R² is alkyl of 1-4 carbons which may besubstituted with from 0-3 halogens. Steps e, f, g, h and i are novel perse while compounds VI, VII, VIII, and IX are also novel.

PREFERRED EMBODIMENTS

The process of this invention is broadly described in the followingterms

a. forming a lower alkyl-6-oxohexanoate by ozonization of a loweralkoxycyclohexene;

b. forming a lower alkyl ester of 7-nitro-6-hydroxy-heptanoic acid byreacting nitromethane with the alkyl-6-oxohexanoate from step a;

c. forming a lower alkyl ester of 7-nitro-6-acyloxyheptanoic acid byreacting the product from step b with an acylating agent;

d. forming a lower alkyl ester of 7-nitro-hept-6-enoic acid;

e. forming a lower alkyl ester ofdl-7-thia-6-nitro-methyl-9-nitro-nonanoic acid by the addition ofnitroethanethiol at position 6 of the reaction product from step dabove;

f. forming dl-6(4-carboalkoxy-butyl)-4H-6H-thieno-[3,4-d]furoxan by theappropriate cyclization reaction;

g. formingdl-2(4-carboalkoxy-butyl)-3,4-bis(alkamido)-2,5-dihydro-thiophene byreduction of the furoxan ring and reaction with an appropriate acylatingagent;

h. forming dl-cis-2(4-carboalkoxy-butyl)-3,4-cis-bis(alkamido)-tetrahydrothiophene by selective hydrogenation;

i. forming dl-biotin by appropriate hydrolysis and cyclizationreactions; and

j. optionally resolving dl-biotin to d-biotin.

Each of the individual process steps will be discussed in a morecomplete manner hereafter pointing out the importance of each step aswell as the novelty of the steps or the intermediate products whereapplicable. It is to be understood that this total synthesis is anentirely novel approach to the preparation of biotin and it is notintended to limit the manner in which each of the compounds are formedin each of the steps. Any method may be used which is suitable for theparticular intermediate.

In the following discussion the Roman numerals refer to the structuresso indicated in the previous reaction scheme.

a. Formation of lower alkyl ester of 6-oxohexanoic acid

The starting point for the overall synthesis of biotin by the process ofthis invention is a lower alkoxy cyclohexene (I) which may be obtainedfrom any supply house which carries the substance or it may be preparedaccording to the process described by D. G. Lidsay, TET, 21, 1673(1965). By lower alkyl is meant an aliphatic hydrocarbon containing 1 to4 carbons and exemplified by methyl, ethyl, propyl, isopropyl, butyl,tertiary butyl, and the like. Preferably the starting material ismethoxycyclohexene and this material shall be referred to hereafter asillustrative but not limiting.

The first step involves ozonizing the double bond of the cyclohexenering to sever the ring and add oxo components at the previously joinedcarbons. The structure obtained is shown as II in the reaction schemepreviously set forth. The methoxycyclohexene is treated with ozone in asuitable solvent under conditions sufficient to give the desiredproduct. Generally these conditions will be a low temperature, that isless than -50° C but no lower than about -150° C. Preferably thetemperature will be between about -75° and -80° C.

Any solvent which is substantially inert to the ozone at thetemperatures employed may be used for this reaction. Because of itsavailability and cheapness, methanol is particularly preferred.Generally about 1 part by weight of methoxycyclohexene will be dissolvedin about 5 to 15 parts by volume of the solvent. In the case of methanola suitable ratio is about 1 part by weight to 10 parts by volume of thesolvent.

The reaction generally takes place merely by placing the reactant andthe solvent in a suitable container, cooling to the temperature desired,then bubbling ozone through the stirred solution for a time sufficientfor the reaction to take place. Generally this time period is no morethan an hour and preferably will be less than about 30 minutes. Excessozone is removed from the reaction solution by an appropriate meanswhich may simply involve bubbling nitrogen through the reactionsolution.

It is thought that an intermediate ozonide may be formed prior to thecomplete severence of the cyclohexene ring. To ultimately form thedesired 6-oxohexanoate the ozonide is treated, e.g. with a suitableamount of dimethylsulfide at the reaction temperature for a short periodof time, for example less than an hour and preferably less than 15minutes, whereafter the solution is allowed to come to ambienttemperature while constantly stirring. This completes the reaction andhastens the formation of the 6-oxohexanoate. Excess solvent is thenremoved by means known in the art such as using a rotary evaporator. Thereaction product, the methyl ester of 6-oxohexanoic acid may then beseparated from the reaction mixture by any means known in the artsufficient to attain such separation, for example distillation.

b. Formation of lower alkyl ester of 7-nitro-6-hydroxy-heptanoic acid

In the next step the alkyl ester of 6-oxohexanoic acid from the previousstep is reacted with nitromethane under basic conditions in a suitablesolvent at low temperatures to form the lower alkyl ester of7-nitro-6-hydroxy-heptanoic acid. Generally the solvent which may beemployed is any solvent which is suitable for performing anitromethylation and hydroxylation as is desired. Because of itseffectiveness, availability and low cost methanol has been found to beparticularly suitable and thus is preferred. Enough nitromethane and asuitable base such as a metal hydroxide is employed to provide thedesired stoichiometric amount of the nitromethyl moiety. A suitable basemay include metal hydroxides such as alkali or alkaline earth hydroxidessuch as the hydroxides of calcium, magnesium, sodium, potassium, and thelike. Sodium hydroxide is particularly well suited for this purpose.Generally only a catalytic amount of base is necessary while a slightmolar excess of nitromethane over the hexanoate will be used. The ratioof nitromethane to hexanoate will be about 1.1:1 to 2.0:1, preferablyabout 1.2:1 to 1.5:1.

Enough solvent is used to thoroughly dissolve the nitromethane and thehexanoate (II) as well as the base and to carry out the reactioneffectively. For example, about 1 part by weight of the reactants willbe dissolved in about 10 parts by volume to 20 parts by volume of thesolvent. The reactants and the base are dissolved in the solvent andmixed together with vigorous stirring at a suitable temperaturegenerally less than 10° C down to -50° C and preferably will be about-5° to +5° C. The reaction is carried on at low temperatures for lessthan an hour, preferably about 30 minutes then is allowed to warm up toambient temperatures. Additional nitromethane may be added at this pointto insure complete reaction. The reaction is allowed to go to completionat room temperature for a suitable period of time generally less than 24hours and preferably will be less than about 12 hours. The resultingproduct is a mixture of the 2 stereo isomers of the methyl ester of7-nitro-6-hydroxy-heptanoic acid, the 6 carbon atom being the asymmetriccarbon. The product is recovered from the reaction mixture usingsuitable means of separation such as solvent removal by rotaryevaporator and the mixture of isomers is then used for the next reactionstep.

c. Forming the lower alkyl ester of 7-nitro-6-acyloxyheptanoic acid

The reaction product from the previous reaction step which includes thetwo stereo isomers, is reacted with a suitable acylating agent to add anacyloxy group at the 6 position of the heptanoic acid chain. The isomersfrom the previous step need not be separated since in the next step adouble bond is formed which is opened at the subsequent step. A suitableacylating agent, such as a lower alkyl (1-4 carbons) acid or its halideor anhydride derivative may be any which are known in the art, but loweralkyl acid anhydrides (e.g. acetic anhydride) have been found to beparticularly valuable. The crude mixture of isomers from step (b) may betaken up in e.g. acetic anhydride and the acetic anhydride is used notonly as a solvent but also as a reactant. Generally about 1 part byweight of the isomer mixture will be dissolved in from 5 to 10 parts byvolume of the anhydride. A small amount of a suitable acid catalyst suchas concentrated sulfuric acid is added and the reactants are allowed toreact in a stirred container at temperatures of about 10°-40° C,preferably about 25° C for a suitable length of time. Generally thelength of time will be no more than 24 hours and preferably will be lessthan about 12 hours. The reaction yields the lower alkyl ester of7-nitro-6-acyloxy-heptanoic acid which may be recovered from thereaction mixture by any suitable method, which includes removal of theexcess anhydride at reduced pressure using a rotary evaporator. Theresulting heptanoate is again a mixture of isomers which need not beseparated before going on to the next step.

d. Formation of the lower alkyl ester of 7-nitro-hept-6-enoic acid

In the next step the product from the previous step is subjected toconditions which are suitable for an elimination reaction to take placeand from a double bond between the 6 and the 7 position. Thus an acyloxyis eliminated and a hept-6-enoic acid is formed.

The elimination reaction will take place in a suitable solvent underbasic conditions at slightly raised temperatures preferably in an inertatmosphere. A suitable solvent is found to be ethyl acetate, (althoughany functional equivalent may be acceptable) which is made slightlybasic by the addition of a small amount of a suitable base, e.g. sodiumbicarbonate, and a catalytic amount of water. Generally about 10 to 20parts by volume of ethyl acetate will be used per one part by weight ofthe reactant and about a half part by weight of the sodium bicarbonate.A catalystic amount of water is that amount of water necessary to causethe reaction to go smoothly. Generally this will be no more than a fewdrops of water in the reaction mixture. The temperature at which thereaction may be run may be anywhere from 25° to 75° C and preferablywill be about 40°-50° C for no more than about 5 hours, preferably lessthan 4 hours. After suitable recovery treatment the lower alkyl ester of7-nitro-hept-6-enoic acid is obtained for the next step.

e. Formation of the lower alkyl ester ofdl-7-thia-6-nitromethyl-9-nitro-nonanoic acid

This step is carried out under conditions suitable for the nucleophilicaddition of a nitroethanethio group at the 6 position of the lower alkylester of the 7-nitro-hept-6-enoic acid. The reaction is carried out in asuitable solvent by dissolving both the nitroethanethiol and thereaction product from the previous step and stirring in an inertatmosphere for a suitable period of time at ambient temperatures. Thereaction is substantially complete after less than 5 hours andpreferably less than about 2 hours. A suitable solvent for this reactionmay be any inert solvent useful for nucleophilic addition reactions,methanol being particularly effective. Methanol is used to about 1 to 10parts by volume of methanol for each part by weight of nitroethanethiol.

f. Formation of dl-6(4-carbo lower alkoxy-butyl)-4H-6H-thieno[3,4-d]furoxan

This step involves the formation of the previously undescribed thieno[3,4-d] furoxan ring by a unique method heretofore unknown in the art.In this method the reaction product from the previous step, that is, thelower alkyl ester of dl-7-thia-6-nitromethyl-9-nitro-nonanoic acid, isplaced in a suitable solvent and reactively contacted with a catalyticamount of a tertiary alkyl amine and phosphorus oxychloride (POCl₃)under substantially anhydrous conditions and in a substantially inertatmosphere. Suitable tertiary alkyl amine catalysts include triethylamine, trimethyl amine, tributyl amine, tripropyl amine, and the like.Suitable solvents for this reaction are inert non-polar, aproticsolvents, e.g. halogenated hydrocarbons such as chloroform, the solventbeing present in very large excess, for example about 10 to 1000 timesthe amount of the reactants used. The reaction is run at ambienttemperatures, that is from about 10° C to about 40° C. Preferably atabout 25° C. Preferably the tertiary alkyl amine catalyst and thephosphorus oxychloride are dissolved in the solvent and a solution ofthe product from the previous step in the solvent is added to themixture. The reaction may take place over a period of anywhere from 10to 24 hours but generally will take place within 18 hours. The resultingdl-6(4-carbo lower alkoxy-butyl)-4H-6H thieno[3,4-d]furoxan is a novelmixture, as well as each of the stereo isomers alone. Although furoxanrings are known to be prepared by the reaction of nitroethane andphenylisocyonate, see for instance JACS, 82, 5339-5342, Oct. 20, 1960"The Reactions of Primary Nitro Paraffins With Nitro Isocyanates",Mukalyama & Hoshino, the preparation of the bicyclic intermediate ofthis invention in a single step from an acyclic precursor usingphosphorus oxychloride and a tertiary amine catalyst is completelynovel. The resulting furoxan compound is substantially stable and may berecovered from the reaction mixture using usual recovery techniques.

g. Formation of dl-2(4-carbo-loweralkoxy-butyl)-3,4-bis(alkamido)-2,5-dihydro thiophene

The novel compound prepared in the previous step may then be convertedinto another novel compound heretofore unknown in the prior art byreducing the furoxan ring and reacting the resulting product with asuitable acylating agent. The furoxan ring may be reduced by catalytichydrogenation or by using a suitable reducing reagent.

In the case of catalytic hydrogenation of the furoxan system, ordinarytemperatures, for example about 10°-100° C, and pressures, of about15-75 psi, are employed in a suitable, inert, liquid solvent, such as alower alkyl alcohol of 1-6 carbon atoms, e.g. methanol, ethanol,isopropanol, n-butanol, and the like. Methanol is particularlyeffective. The catalyst may be any suitable platinum metal catalystwhich is generally effective for hydrogenating a furoxan ring;especially effective is a palladium catalyst. The catalyst may besupported or unsupported, but preferably is supported on a carbonsupport. It appears that the hydrogenation medium should be slightlyacidic to nullify any inhibitory effects which may occur due to amineintermediates. A trace amount of perchloric acid has been found to beparticularly effective. The reduced product is then acylated with anacylating agent such as a suitable carboxylic acid of 1-4 carbon atomsor derivatives such as an acid chloride or anhydride, and may besubstituted with 0-3 halogenatoms and preferably is an acetic acid orderivative such as the acylating agent trifluoroacetic anhydride.

Alternatively, the furoxan ring is reduced by reactively contacting thecompound prepared in the previous step with a suitable reagent, e.g. afreshly activated zinc powder or, preferably, a zinc-silver couple,which is sufficient for furoxan reduction. Freshly activated zinc may bereadily prepared by treating zinc dust with 10% hydrochloric acid over ashort period of time with concentrated hydrochloric acid being added tomaintain a vigorous evolution of hydrogen. The resulting solid issubsequently washed with water, acetone, ether, and hot acetic acid. Thezinc-silver couple is prepared by thereafter treating the activated zincwith hot glacial acetic acid containing silver acetate for a shortperiod of time. The resulting dark catalyst may be washed with drymethoxyethane to yield a suitable zinc-silver couple.

Preferably, in the reduction using activated zinc or the zinc-silvercouple, the novel furoxan from step (f) is treated to simultaneouslyreduce the furoxan and acylate the amine moieties of the resultingreduced thiophene ring. This may be accomplished by placing the furoxanin trifluoroacetic anhydride and optionally a suitable inert, oxygenatedhydrocarbon solvent such as dimethoxyethane and placing the solution inreactive contact with the activated zinc or zinc-silver couple. Themixture is kept in constant motion by stirring and the reaction takesplace at low temperatures of about -5° to +10° C, preferably about 0° to5° C over a period of time of about 2 hours preferably less than an hourand a half. The resulting reaction product can be recovered from thereaction mixture by usual recovery methods and separated, for example,on a silica gel column using chloroform as an eluent. The resultingcompounds, 2(4-carbo loweralkoxy-butyl)-3,4-bis(alkamido)-2,5-dihydrothiophene are novel, eitheras a mixture of the dl-isomers or either isomer alone as, is thisparticular step for the preparation of the novel compounds.

h. Formation of dl-cis-2(4-carbo loweralkoxy-butyl)-3,4-cis-bis(alkamido)-tetrahydrothiophene

The novel dihydrothiophene from the previous step is hydrogenated usinga suitable hydrogenation catalyst to form the novel compound of thisstep. The resulting hydrogenation products are novel compoundsheretofore unknow in the art and the process for preparing saidcompounds is also novel. The hydrogenation is carried out in a suitablesolvent using a hydrogenation catalyst suitable for effecting theselective hydrogenation of the thiophene ring. A particularly valuablecatalyst is a palladium hydroxide catalyst which is approximately 20%palladium on G-60-charcoal, prepared according to a process described byM. Pearlman, Tetrahedron Letters, 1967, 1663. Preferably the catalyst isprehydrogenated. The hydrogenation is carried out in a suitablehydrogenating apparatus such as a Parr apparatus at suitablehydrogenating temperatures and pressures, for example a pressure of 50to 70 pounds per square inch is usable and preferably 60 pounds persquare inch is optimum. The temperature may be anywhere from 0 to 50°but preferably will be about room temperature, that is about 25° C. Thereaction may take anywhere from 12 to 24 hours but preferably will be 24hours or more to effect complete reaction. Preferably, the hydrogenationsolvent is methanol. The cis-2(4-carbo lower alkoxy-butyl)-3,4-cis-bis(lower alkamido)-tetrahydrothiophenes are novel compounds either asmixtures of the dl-isomers or as the d- or 1- isomer alone.

i. Formation of dl-biotin

The novel saturated thiophene from the previous step is converted tobiotin by removing the acyl moieties and acylative cyclization of theresulting diamine. The 2 steps may be carried out without isolation ofintermediates by treating the tetrahydrothiophene from the previous stepwith phosgene at low temperatures in a suitable solvent in the presenceof a mild base. A suitable solvent may be a mixture of methanol andbenzene, the methanol being substantially completely deoxygenated. Thereaction mixture is stirred for several hours preferably no more than 2and then is treated to recover the biotin.

j. Formation of d-biotin

The dl-biotin may then be resolved to obtain the d-biotin which is theactive substance. This may be done by any method known in the art.

The following examples are given to illustrate the total synthesis ofbiotin as well as the synthesis of the unique intermediates in thepreparation of biotin and the novel individual steps for preparingbiotin intermediates. The examples are representative of the conditionsunder which biotin and the intermediates may be prepared but is not tobe read in a limiting sense.

EXAMPLE 1 Preparation of Biotin

General Discussion of Experimental Parameters, Procedures, and Analysis

General Workup: In each step, the crude reaction product mixture wastaken up in ethyl acetate, washed with aqueous solutions as indicated inthe respective steps hereafter or saturated sodium chloride solution.The organic layer was dried over magnesium sulfate and evaporated usinga laboratory rotary evaporator at the temperature indicated or at about30° C.

Preparative column chromatography: As absorbent silica gel by Merck(particle size 0.05-0.2 mm) was used in 80-fold amount, and the solventsused are shown below where appropriate.

Thin Layer Chromatography (TLC): Analtech "Uniplate" precoated silicagel plates were used. Detection of the TL-spots was effected underUV-lamp and with ammonium molybdate-water-sulfuric acid solution (MOLLY)followed by heating the plate.

Crystallization were carried out from acetone-hexane or the solventsindicated in the steps hereinafter.

Melting points were obtained in open capillaries in an oil bath and arenot corrected.

IR-spectra were run on a Perkin-Elmer 237 B Instrument from 2-3%substance/CHCl₃ -solutions or specified conditions. The absorption bandsare given in wave numbers (cm-¹).

The UV.-spectra were determined in ethanol solutions on a Carry Model 14Recording Spectrophotometer. λ max.-terms are in nm and the intensitiesquoted as ε.

The mass spectras were recorded on Atlas CH-4 or CH-7 instruments.

The 'H-nmr-spectras were run on Varian instruments HA-100 (100MHZ) andVarian A-60(60MHZ). The chemical shifts given in δ-terms (ppm) withtertramethylsilane (TMS) as internal standard (=0). AbbreviationsS=singlet, d=doublet, t=triplet, q=quadruplet, m=multiplet, b=broadsingle, J=coupling constant in HZ. The assigned number of protons comesfrom the electronically calculated integration and corresponds with thechemical structure.

The ¹³ C-nmr-spectras were recorded on a Bruker C13 spectrometer22.63MHZ.

PROCESS

In the following discussion the Roman numerals refer to the reactionscheme under "Summary of the Invention".

a. Forming Methyl-6-oxohexanoate (II-R=CH₃)

1.12 g (0.01 mol) methoxycyclohexene (I, prepared according to D.G.Linsay, Tet, 21, 1973 (1965),) were dissolved in 10 ml methanol, cooledto -78° C then ozonized for 30 min. The excess of ozone was removed withnitrogen. The ozonide in solution was treated with 800 mg dimethylsulfide over a period of 15 min. at -78°. The solution was stirred foranother 30 min. and then warmed up to room temperature. After stirringover night, the solvent was removed using a rotary evaporator (bathtemperature below 40°). Distillation of the reaction mixture yielded 912mg (63%) of methyl-6-oxohexanoate (II) as an oil, b.p. 60° (0.1mm).

ir (CCl₄): 2970, 2820, 2720, 1740, 1730.

nmr (A-60): 1.5-1.8, m, CH₂ (3&4); 2.2-2.6, m, CH₂ (2&5); 3.66, S,COOCH₃ ; 9.7, m, CHO.

¹³ C-nmr: 21.5, C (4); 24.3, C (3); 33.6, C (2); 43.4, C (5); 51.4,OCH₃, 173.1, C (6); 201.4, C (1).

ms m/e 145 (M³⁰ +H); 116 (M⁺ --CO); 113 (M⁺ --OCH₃); 101 (M⁺ --CHOCH₂);87; 74.

b. Forming methyl-7-nitro-6-hydroxy-heptanoate (III-R=CH₃)

To a solution of 15.3 g nitromethane and 240 mg sodium hydroxide in 350ml methanol a solution of 28.8 g aldehydoester II in 150 ml methanol wasadded at 0° C with vigorous stirring. The stirring was continued for 30min. at 0° C. The reaction mixture was allowed to warm up to roomtemperature and another 5 ml nitromethane was added and the mixturestirred overnight at room temperature. General workup as discussed aboveyielded 36.6 g (89%) of methyl-7-nitro-6-hydroxy-heptanoate (III) as ayellow oil (mixture of two isomers).

nmr: 1.3-1.8, m, CH₂ (3, 4&5); 2.2-2.5, m, CH₂ (2); 3.68, S, COOCH₃,4.4, m, --CH₂ --NO₂.

c. Forming methyl-7-nitro-6-acetoxy-heptanoate (IV-R=CH₃ ; R¹ =CH₃

The crude mixture of isomers from b (III) was taken up in 280 ml aceticanhydride. 1.0 ml conc. H₂ SO₄ was added and the reaction mixture wasstirred overnight at room temperature. After removal of acetic anhydrideat reduced pressure, the reaction mixture was treated according to thegeneral workup, yielding 43.7 g (99%) of a crude brown oil consisting ofthe isomers of methyl-7-nitro-6-acetoxy-heptanoate (IV).

d. Forming the methyl ester of 7-nitro-hept-6-enoic acid (V-R=CH₃)

22 g crude nitroacetate (IV) were dissolved in 400 ml ethyl acetate and11 g sodium bicarbonate and 20 drops water were added. The mixture wasstirred 4 hours at 50° under nitrogen. The solution was filtered; theresidue washed with ethyl acetate, dissolved in water and extracted withethyl acetate. The combined organic layers were washed with water, driedand evaporated. After column chromatography with chloroform onsilicagel, 6.3 g (38%) of the methyl ester of 7-nitro-hept-6-enoic acid(V) was collected (33% referred to II).

ir: 1735, 1645, 1520, 1345.-uv (εtoh) max 265 nm (8700).-

nmr: 1.4-1.7, m,. CH₂ (3&4); 2.1-2.4, m, CH₂ (2&5); 2.68, s, COOCH₃ ;6.8-7.6, CH (6&7).-

¹³ C-nmr: 24.3, C (3); 27.2 & 28.1, C (4&5); 33.5, C (2); 51.6, OCH₃ ;140.0 & 142.1, C (6&7); 173.7, C (1).

Calculated for C₈ H₁₃ NO₄ : C..51.33%; H..7.00%; N..7.48% Found for V:C..51.21%; H..7.02%; N..7.16%

e. Forming the methyl ester of dl-7-thia-6-nitromethyl-9-nitro-nonanoicacid (VI-R=CH₃)

4.0 g nitroethyl thiol (prepared according to Example 2) were dissolvedin 8 ml methanol, the ester (V) from the preceeding step in 8 mlmethanol was added, and the mixture was stirred under N₂ for 2 hours atroom temperature. General workup gave 7.5 g (80%)dl-7-thia-6-nitromethyl-9-nitro-nonanoic acid methylester (VI) as ayellow oil.

ir: 1725, 1550, 1435.

ms: 263 (M⁺ --CH₃ O); 248 (M⁺ --NO₂)

Calc. for C₁₀ H₁₈ N₂ O₆ S: C..40.81%; H6.17%, N9.52% Found for C₁₀ H₁₈N₂ O₆ S: C..40.73%; H6.23%; N9.47%

f. Forming dl-6(4-carbomethoxy-butyl)-4H-6H-thieno[3,4-d]furoxan(VII-R=CH₃ ; R² =CF₃)

12.4 g phosphorus oxychloxide (POCl₃) were dissolved in 400 mlchloroform (dried over molecular sieve) and 20.1 g triethylamine(freshly distilled over LiAlH₄) were added. The mixture was allowed tocool to room temperature and 2.94 g of the ester VI from step f in 200ml dry CHCl₃ were added over a period of 18 hours at room temperatureunder N₂. The mixture was stirred an additional 11/2 hours at roomtemperature. The black reaction mixture was poured on ice containing 5ml conc. HCL, the layers separated, and the water phase washed threetimes with chloroform. The combined chloroform layers were washed with asmall portion of saturated NaCl-solution, dried and evaporated using arotary evaporator (water bath below 40° C). The black tar waschromatographed over a short column (20 g silicagel) with ether: hexane(2:1) as eluant yielding 2.1 g (81%) of the two isomeric furoxans (VII).

uv: 234, 265 nm (1750, 5130);

ir 1735, 1645, 1455, 980;

nmr: 1.5-1.8, n, CH₂ (3, 4&5); 2.2-2.4, m, CH₂ (2); 3.66, s, OCH₃ ; 3.9,d, CH₂ (9), gem=12H. 4.4, b, CH (6);

¹³ C-nmr: shows two isomeric compounds

ms: 242 (M⁺ --O), 227 (M⁺ --OCH₃).

Calculated for C₁₀ H₁₄ N₂ O₄ S: C-46.50% H-5.46% N-10.85%

Found for C₁₀ H₁₄ N₂ O₄ S: C-46.56%; H-5.77%; N-10.53%

g. Forming dl-2 (4carbomethoxy-butyl)-3,4-bis-(trifluoroacetamido)-2,5-dihydro-thiophene(VIII-R=CH₃ ; R² =CF₃)

Preparing a zinc-silver catalyst

17 g zinc dust (analytical grade) were treated with 100 ml 10% HCl andconc. HCL added to maintain a vigorous evolution of hydrogen over 10minutes. The remaining solid was subsequently washed with two 100 mlportions of water, two 100 ml portions acetone, and two 100 ml portionsether, followed by washing with 100 ml glacial acetic acid (hot, warmedon the steam bath for 15 min). A second 100 ml hot glac. acetic acidcontaining 200 mg silver acetate (analytical grade) was added. Themixture was vigorously shaken for 1 minute, the dark catalyst thenwashed with 2 × 100 ml dry dimethoxyethane (DME), yielding a zinc/silvercatalyst sufficient for furoxan reduction. Proof of the activity of thecatalyst was evident due to a rise of temperature as thefuroxan/DME/(CF₃ CO)₂ O mixture was added.

Reduction of Furoxan and reaction with trifluoroacetic acid

2.5 g of the furoxan (VII) in 16 ml dry dimethoxyethane and 16 mltrifluoroacetic anhydride were added to the catalyst (prepared from 17 gZn-dust as discussed above) in 20 ml dimethoxyethane at 5° C over 75minutes. The stirring was continued for 10 minutes. General workup gave1.980 g of a yellow oil containing impurities (complex GLC). Separationon 300 g silica gel with chloroform containing 1% MeOH yielded 1.59 g ofthe pure dihydrothiophene (VIII) (38%) as white needles; mp 84°-85°(ether).

ir: 3250, 1735, 1710, 1170, 880;

nmr: 1.4-1.8, m, CH₂ (3,485); 2.25-2.45, m, CH₂ (2); 3.65, S, OCH₃ ;3.85, m, CH (a), 4.1, m, CH (9); 4, 4, b, CH (6);

¹³ C-nmr: 24.12, C (3); 25.45, C (5); 32.74, C (4); 33.81, C (2) 34.92,C (9); 48.60, C (6); 51.79, OCH₃ ; 109.26 & 121.98, CF₃ ; 124.12, C (8);125.3, C (7); 174.84, C (1).

ms: 422 (M⁺), 391 (M⁺ --OCH₃), 309 (M⁺ --NH₂ COCF₃), 307 (M⁺ -- sidechain).

Calculated for C₁₄ H₆₁ N₂ O₄ F₆ S: C, 39.81%; H, 3.82% N, 6.63%.

Found for C₁₄ H₁₆ N₂ O₄ F₆ S: C, 40.02% H, 3.96%, N, 6.77%.

h. Forming dl-cis-2 (4-carbomethoxy-butyl)-3,4-cis-bis(trifluoroacetamido)-tetrahydrohiophene (IX-R=CH₃ ; R² =CF₃)

120 mg of the dihydrothiophene (VIII) were dissolved in 2 ml MeOH andadded to a suspension of 120 mg prehydrogenated (1 hour, 60 psi) Pd(OH)₂ -catalyst (ca.20% Pd on DarcoG-60-charcoal, prepared according toM. Pearlman, Tetrahedron Letters, 1967, 1663.) in 2 ml MeOH. Thehydrogenation was carried out in a Parr-apparatus with a small bottle,at 60 psi H₂ and room temperature, over 24 hours. The catalyst wasfiltered off and washed with methylene chloride. The solvent wasevaporated yielding 84 mg. of the tetrahydrothiophene (IX) as an oilwhich decomposed during all purificication attempts, and was thus useddirectly for the next step.

gc-ms: 393 (M⁺ --OCH₃), 311 (M⁺ -NH₂ COCF₃), 197 (M⁺ --2NH₂ COCF₃), 166(M⁺ --2NH₂ COCF₃ --OCH₃).

i. Forming dl-biotin (X)

The 84 mg hydrogenation product (IX) were dissolved in 10 mldeoxygenated methanol and 1 g K₂ CO₃ in 10 ml H₂ O was added. Thereaction mixture was stirred overnight at room temperature under N₂.Then 40 ml phosgene (12.5%) in benzene was added to the precooled (0°)mixture. After stirring for 2 hours at room temperature the reaction wastaken up in ethyl acetate and 10% acetic acid, washed with a smallamount of ice water and filtered with about 1 g Darco G-60 to remove theyellow color. The charcoal was well washed and then eluted with 5 ml 5 Nammonium hydroxide. The solvents were evaporated and the product mixturechromatographed on silica gel with ethyl acetate and 5% acetic acidyielding 53 mg of dl-biotin (X) which, after recrystallization from 5 Nammonium hydroxide (solution and reacidification) melted at 224°;

ir: 3350, 3250, 3060, 1705, 1660, 1250, 1180;

nmr: 1.25-1.70, m, CH₂ (3, 4&5); 2.10-2.30, m, CH₂, m, CH₂ (2); 2.6, D,CH (9β); Jaβ,αx=12 HZ; 2.85, dd, CH (9x), ---- J₉ α,₈ =5HZ; 3.1-3.2, m,CH (6); 4.1-4.4, m, CH (7&8); 5.3-5.5, b, NH (2H).-¹³ C

nmr (DMSO): 24.48, C (3); 28.02, C (4&5);33.45, C (2); 39.53, C (9);55.33, C (6); 5 g.20, C (8); 61.05, C (7); 162.77, N₂ C=O; 174.45, C(1).

ms: 244 (M⁺); 184 (M⁺ --CH₂ COOH-H); 112;97 (base peak); 85; identicalwith authentic d-biotin ms.

j. Resolution of dl-biotin

A mixture of 7 mg dl-biotin (14) and 7 mg (ca. 10% excess) of L(+)-arginine was dissolved in 1 ml H₂ O and the solution was dilutedwith isopropyl alcohol. The solution was cooled to 0° C and kept in therefrigerator overnight. The crystals were filtered and washed withacetone yielding 6.5 mg. The L (+)-arginine salt of dl-biotin wasdissolved in 1 ml H₂ O and acifified with 1N HCL. The crystallined-biotin was filtered, washed with cold water and dried in vacuoyielding 1.8 mg of d-biotin (27% ) m.p. 228°-229° C. A mixed m.p. withd-biotin gave no depression in m.p.

EXAMPLE 2 Preparation of Nitroethanethiol

This example shows a particularly preferred method of preparingnitroethanethiol which is used in step (e), above. 80 g dryparaformaldehyde were suspended in 1.6 l nitromethane (CH₃ NO₂). Afteraddition of 3 ml of 3 N potassium hydroxide (KOH) in methanol thesuspension was stirred for 30 min. The now clear solution was acidifiedwith 1.5 ml conc. sulfuric acid, stirred for an additional 30 min andfiltered. The excess CH₃ NO₂ was distilled off at 30°-50° C bathtemperature and the residue distilled at about 1 mm/50° C yielding 106 gcolorless nitroethanol.

64 g nitroethanol, 50 ml glacial acetic acid, 250 ml benzene and 5 dropsconc. H₂ SO₄ were refluxed, using a water separator for 8 hours; 12 mlof H₂ O were collected. The solution was evaporated and vacuum distilled(1mm at 60° C) yielding 89 g colorless nitroethylacetate.

80 g sodium hydroxide were dissolved in 500 ml water and 35 mlphosphorous thiochloride (PSCl₃ --98%) were added to maintain thereaction temperature between 75° and 85° C (ca. 30 min). The reactionwas stirred at 80° C until all the PSCL₃ had reacted (45 min). Then thereaction mixture was put into the refrigerator (0° C) over night. Theprecipitated white crystals were filtered off and washed with 100 mlEtOH. The crystals were dissolved in 250 ml distilled water at 45° C andreprecipitated by the slow addition of 200 ml ethanol (EtOH) under rapidstirring. The solution was cooled to room temperature, filtered, and thecollected white crystals were washed with 100 ml EtOH.

The resulting product was dehydrated by stirring in 600 ml absolute MeOHfor 11/2 hours. The fine white powder was filtered off and dried at 105°C (vacuum) for 1 hour yielding 38 g (59%) of anhydrous trisodiumthiophosphate (Na₃ PO₃ S). To a solution of 54.3 g Na₃ PO₃ S in 600 mldist. water were added 0.5 g benzyltriethylammonium chloride, themixture cooled to 15° and then 39.9 g nitroethyl acetate, preparedabove, was added during 15 min, the temperature being maintained below20° C. The mixture was stirred at room temperature for 16 hours, thenacidified with 60 ml conc. HCl and hydrolyzed at 40° for 1 hour. After awork up according to the general procedure in Example I, the residue wasdistilled yielding 9.7 g (30%) nitroethanethiol b.p. 37°-39° (0.3 mm).

We claim as our invention:
 1. The process of preparing biotin whichcomprisesa. ozonizing a lower alkoxycyclohexene to form the lower alkylester of 6-oxohexanoic acid; b. reacting nitromethane with the loweralkyl ester of 6-oxo-hexanoic acid from step (a) under basic conditionsin the presence of a solvent suitable for nitromethylation to form thelower alkyl ester of 7-nitro-6-hydroxy-heptanoic acid; c. reacting theproduct of step (b) with a lower alkanoic acid or its halide oranhydride derivative to form the lower alkyl ester of 7-nitro-6-loweralkanoyl -heptanoic acid; d. placing the reaction product of step (c) ina suitable solvent under basic conditions at slightly raisedtemperatures to form the lower alkyl ester of 7-nitrohept-6-enoic acid;e. reacting nitroethanethiol with the reaction product from step (d) inan inert atmosphere, in a solvent and under conditions suitable fornucleophilic addition to form the lower alkyl ester ofdl-7-thio-6-nitromethyl-9-nitrononanoic acid; f. reactively contactingthe reaction product of step (e) with a catalytic amount of a tertiaryalkyl amine and phosphorus oxychloride under substantial anhydrousconditions in a substantially inert atmosphere in a suitable solvent toform dl-6-(4-carbo lower alkoxy-butyl)-4H-6H-thieno [3,4-d]-furoxan; g.reducing the product from step (f) by catalytic hydrogenation or byusing a suitable reducing agent and acylating with a suitable carboxylicacid of one to four carbon atoms or its acid chloride or anhydridederivative, said acid or its derivative being substituted with from 0 to3 halogens, to form dl-2(4-carbo loweralkoxy-butyl)-3,4-bis(alkamido)-2,5-dihydro-thiophene; h. selectivelyhydrogenating the product from step (g) to form dl-cis-2(4-carbo loweralkoxy-butyl)-3,4-cis-bis(alkamido)-tertrahydro-thiophene; i.hydrolyzing and cyclizing the product from step (h) to form dl-biotin.2. The process of claim 1 wherein said dl-biotin is resolved to form dbiotin.
 3. The process of claim 1 wherein said lower alkoxyclohexene ismethoxycyclohexene and said acylating agent in step g is trifluoroaceticanhydride.